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determination of the protein concentration of a cell extract

Using the Bradford Method

Abstract

Dilutions of the 1.00mg/cm3 BSA protein stock solution were prepared, to make, 0.00, 0.20, 0.40, 0.60 and 0.80 mg/cm3 solutions and treated with Bradford reagent. Replicates of each of the samples were made at the same time. Absorbance was measured for each which gave 0.000, 0.161, 0.347, 0.445, 0.552 and 0.783 respectively. A calibration curve was constructed using these values. The absorbance for the cell extract (undiluted) and for its 0.5 dilution was found; 0.542 and 0.279 respectively, which were extrapolated on the calibration graph to find concentration values of 0.69 mg/cm3 and 0.37 mg/cm3. The initial concentration of the diluted sample of extract was calculated to be 0.74 mg/cm3, and the undiluted sample 0.69 mg/cm3 using the graph. Value of concentration of cell extract hence is 0.7 mg/cm3 to the nearest decimal place.

Introduction

The Bradford assay is a convenient way of determining cell extract protein concentration, as the reagent used is not affected by most of the reducing agents, solvents, thiols, buffers, salts, and metal chelating agents that may be present in the cell extracts. However it’s compatibility with detergents is very low, hence is not an ideal method for determination of membrane bound proteins, which have to first be solubilised using detergents to extract.(Thermo Scientific, n.d.)

The Bradford assay is a colorimetric protein assay. The Bradford reagent used contained Commassie Blue dye dissolved in a mixture of methanol and phosphoric acid. Under these high acidic conditions Commassie Blue dye is stable at the doubly-protonated red (cationic) form, but it binds to protein gaining it’s most stable un-protonated (anionic) blue form. (Here absorbance maximum [Amax -wavelength at which absorbance is maximum] is shifted from 465 nm to 595 nm with the colour change). .(Thermo Scientific, n.d.)

The assay is based on the formation of the protein-dye complex which leads to the colour change of the dye. It had been discovered that the formation of the colour is related the protein consisting of the amino acids arginine, lysine and histidine.(Thermo Scientific, n.d.)

This colour change is measured in terms of absorption of light by solution containing the complex. The amount of absorption of light is directly proportional to the amount of protein present in the solution. This colour change instant remains unaltered for about 1 hour allowing enough time to take spectrophotometer readings for all samples. .(Thermo Scientific, n.d.)

BSA (Bovine Serum Albumin) is usually used because of its stability; it has no effect in most biochemical reactions and its low cost; because a great amount can be obtained from the purification of bovine blood, a by-product of the cattle industry. .(Thermo Scientific, n.d.)

BSA protein stock solution it diluted at different levels to make a series of dilutions of known concentration. The Bradford assay is performed on them to illustrate the relationship between, concentration of protein and absorbance of light by protein-complex containing solutions. These values are then used to calibrate a standard graph (Graph1) which can be used as a template in the determination of the unknown protein concentration of the cell extract. .(Thermo Scientific, n.d.)

The aim of this experiment was to determine the concentration of protein of a cell extract using the Bradford method.

Materials and Methods

The materials and method used in the experiment are as stated in the Cell biology and Development- practical guide

Replicates of each test-tube were made. They were made and incubated for the same amounts of time.

All test tubes were incubated for: 20 minutes

.

Results

Table 1 shows the values of BSA standard solution concentrations and absorbance values used to construct the calibration graph (Graph 1) below. The graph shows there is a steady increase in the absorbance with the increase in concentration of the BSA standard solutions. The graph shows that there is a direct proportionality between the concentration of BSA protein solution and the absorbance by each sample.

Tube 7 replicates were a 1:1 dilution of the cell extract to make a final volume of 100µl (50µl cell extract + 50µl water). Tube 8 replicates contained 100µl of undiluted cell extract. The concentrations were found out using the graph, by extrapolating the values of absorbance for samples 7 and 8. The concentrations of the cell extract in sample 7 in both replicates are 0.37 (mg/cm3) and that of samples 8 are almost double this concentration; 0.69 (mg/cm3).

Table 1.

Tube number

BSA Standard 1

BSA Standard

2

BSA Standard

3

BSA Standard

4

BSA Standard

5

BSA Standard

6

Unknown cell extract protein

7

Unknown cell extract protein

8

Replicates of each tube

Rep1

Rep2

Rep1

Rep2

Rep1

Rep2

Rep1

Rep2

Rep1

Rep2

Rep1

Rep2

Rep1

Rep2

Rep1

Rep2

Final Concentration of protein in tube (mg/cm3)

0

0

0.20

0.20

0.40

0.40

0.60

0.60

0.80

0.80

1.00

1.00

0.37

0.37

0.69

0.69

Absolute amount of protein in tube (µl)

0

0

20

20

40

40

60

60

80

80

100

100

37

37

69

69

Absorbance (A)

0.000

0.000

0.159

0.162

0.293

0.400

0.440

0.450

0.560

0.543

0.777

0.789

0.280

0.277

0.519

0.564

Average Absorbance of replicates of each tube (A)

0.000

0.161

0.347

0.445

0.552

0.783

0.279

0.542

Table 1. Showing the concentration of BSA standard solutions, corresponding absorbance and the values for the absolute amount of protein in the samples.

Graph 1.

Graph 1. The calibration graph showing the direct proportionality of the final concentration of BSA in tube against Average Absorbance by each set of samples.

Discussion

Since the cell extract in tube 7 was diluted by half the concentration of tube 8 (contained undiluted, cell extract with original concentration), ideally the concentration of cell extract of tube 7 must be twice that of tube 8. But the concentration of the protein of solution in tube 7 is 0.37 (mg/cm3) and that of tube 8 is 0.69 (mg/cm3).

When initial concentration of the cell extract for tube 7 was calculated by using the 0.37(mg/cm3) as the final concentration after dilution, and by taking the dilution factor into consideration, it was found that the initial cell extract concentration was 0.74 (mg/cm3).

Calculation of the initial concentration (of sample in tube 7) by taking dilution factor to consideration:

C1 – Initial concentration

V1 -Initial volume

C2 - Final concentration

V2 – Final volume

C1V1=C2V2

C1x 50 = 0.37 x100

C1= 0.74(mg/cm3) .

The obtained values for the initial concentration (undiluted) of the cell extract using the calibration graph and calculated values are similar; 0.69 (mg/cm3) and 0.74 (mg/cm3), although not the same.

The inaccuracy was due to human errors caused during pipetting. E.g. an unnoticed air bubble sucked in when pipetting out cell extract.

The data labelled 0.35 and 0.55 in the Graph 1.were ignored when drawing the best fitted line across the points. This increased the quality of the calibration graph. The absorbance values for the replicates of tube 3 tube 5 had contrast difference in values. This was due to incorrect pipetting during the experiment. The replicates were not identical.

Limitations of the assay are that dye tends to bind with arginyl and lysyl amino acids more readily than other amino acids of proteins, leading to dissimilarity of results of the assay when performed with various proteins, this is the main disadvantage of the assay.( MolecularStation,n.d.)

In addition, the Bradford reagent used is restrained in the presence of detergents, thus membrane bound proteins cannot be assessed using this method as the extraction of them is done using detergents. Proteins which are low-acid soluble cannot be assessed using this method as the Coomassie dye in the Bradford reagent is extremely acidic.(Thermo Scientific, n.d.)

The linear range of the calibration graph is only around 2 µg/ml to 120 µg/ml. This makes it necessary that dilutions of the sample are made before analysis.(HistoSoft,n.d.)

The purpose of the assay was achieved successfully and the concentration of the cell extract was found to be 0.7 (mg/cm3) to the nearest decimal point.

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